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1.
Braz J Biol ; 83: e248814, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34550286

RESUMO

The karyotype and constitutive heterochromatin pattern of the white stork Ciconia ciconia samples obtained from Manzala lake, Dimiaat, Egypt was described. Somatic cells of Ciconia ciconia samples have diploid number 2n= 68 chromosomes. Out of 68 chromosomes, 11 pairs including sex chromosomes were macrochromosomes and the remaining pairs were microchromosomes. Of the 11 macrochromosome pairs, no.1, 2, 4 and 5 were submetacentric and pairs no. 6, 7 and 8 were described as metacentric. In addition, the autosome pair no.3 was subtelocentric, while autosome pair no.9 was acrocentric. Also, the sex chromosome Z represents the fourth one in size and it was classified as submetacentric while, W chromosome appeared as medium size and was acrocentric. Furthermore, C-banding pattern (constitutive heterochromatin) revealed variation in their sizes and occurrence between macrochromosomes. Pairs no. 7 and 8 of autosomes exhibited unusual distribution of heterochromatin, where they appeared as entirely heterochromatic. This may be related to the origin of sex chromosomes Z and W. However, there is no sufficient evidence illustrate the appearance of entirely heterochromatic autosomes. Therefore, there is no available cytogenetic literature that describes the C-banding and karyotype of Ciconia Ciconia, so the results herein are important and may assist in cytogenetic study and evolutionary pattern of Ciconiiformes.


Assuntos
Heterocromatina , Cromossomos Sexuais , Animais , Aves , Heterocromatina/genética , Cariótipo , Cariotipagem , Cromossomos Sexuais/genética
2.
BMC Bioinformatics ; 22(Suppl 6): 396, 2021 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-34362304

RESUMO

BACKGROUND: Meiotic recombination is a vital biological process playing an essential role in genome's structural and functional dynamics. Genomes exhibit highly various recombination profiles along chromosomes associated with several chromatin states. However, eu-heterochromatin boundaries are not available nor easily provided for non-model organisms, especially for newly sequenced ones. Hence, we miss accurate local recombination rates necessary to address evolutionary questions. RESULTS: Here, we propose an automated computational tool, based on the Marey maps method, allowing to identify heterochromatin boundaries along chromosomes and estimating local recombination rates. Our method, called BREC (heterochromatin Boundaries and RECombination rate estimates) is non-genome-specific, running even on non-model genomes as long as genetic and physical maps are available. BREC is based on pure statistics and is data-driven, implying that good input data quality remains a strong requirement. Therefore, a data pre-processing module (data quality control and cleaning) is provided. Experiments show that BREC handles different markers' density and distribution issues. CONCLUSIONS: BREC's heterochromatin boundaries have been validated with cytological equivalents experimentally generated on the fruit fly Drosophila melanogaster genome, for which BREC returns congruent corresponding values. Also, BREC's recombination rates have been compared with previously reported estimates. Based on the promising results, we believe our tool has the potential to help bring data science into the service of genome biology and evolution. We introduce BREC within an R-package and a Shiny web-based user-friendly application yielding a fast, easy-to-use, and broadly accessible resource. The BREC R-package is available at the GitHub repository https://github.com/GenomeStructureOrganization .


Assuntos
Heterocromatina , Aplicativos Móveis , Animais , Mapeamento Cromossômico , Drosophila melanogaster/genética , Heterocromatina/genética , Recombinação Genética
3.
Methods Mol Biol ; 2351: 307-320, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382197

RESUMO

The transition from silenced heterochromatin to a biologically active state and vice versa is a fundamental part of the implementation of cell type-specific gene expression programs. To reveal structure-function relationships and dissect the underlying mechanisms, experiments that ectopically induce transcription are highly informative. In particular, the approach to perturb chromatin states by recruiting fusions of the catalytically inactive dCas9 protein in a sequence-specific manner to a locus of interest has been used in numerous applications. Here, we describe how this approach can be applied to activate pericentric heterochromatin (PCH) in mouse cells as a prototypic silenced state by providing protocols for the following workflow: (a) Recruitment of dCas9 fusion constructs with the strong transcriptional activator VPR to PCH. (b) Analysis of the resulting changes in chromatin compaction, epigenetic marks, and active transcription by fluorescence microscopy-based readouts. (c) Automated analysis of the resulting images with a set of scripts in the R programming language. Furthermore, we discuss how parameters for chromatin decondensation and active transcription are extracted from these experiments and can be combined with other readouts to gain insights into PCH activation.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Regulação da Expressão Gênica , Heterocromatina/genética , Ativação Transcricional , Animais , Proteína 9 Associada à CRISPR/genética , Cromatina/genética , Cromatina/metabolismo , Fibroblastos/metabolismo , Imunofluorescência/métodos , Expressão Gênica , Heterocromatina/metabolismo , Processamento de Imagem Assistida por Computador , Camundongos , Microscopia de Fluorescência , Ligação Proteica , Transfecção , Fluxo de Trabalho
4.
5.
Prog Mol Subcell Biol ; 60: 145-167, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34386875

RESUMO

Satellite DNAs are tandemly repeated sequences organized in large clusters within (peri)centromeric and/or subtelomeric heterochromatin. However, in many species, satellite DNAs are not restricted to heterochromatin but are also dispersed as short arrays within euchromatin. Such genomic organization together with transcriptional activity seems to be a prerequisite for the gene-modulatory effect of satellite DNAs which was first demonstrated in the beetle Tribolium castaneum upon heat stress. Namely, enrichment of a silent histone mark at euchromatic repeats of a major beetle satellite DNA results in epigenetic silencing of neighboring genes. In addition, human satellite III transcripts induced by heat shock contribute to genome-wide gene silencing, providing protection against stress-induced cell death. Gene silencing mediated by satellite RNA was also shown to be fundamental for the early embryonic development of the mosquito Aedes aegypti. Apart from a physiological role during embryogenesis and heat stress response, activation of satellite DNAs in terms of transcription and proliferation can have an evolutionary impact. Spreading of satellite repeats throughout euchromatin promotes the variation of epigenetic landscapes and gene expression diversity, contributing to the evolution of gene regulatory networks and to genome adaptation in fluctuating environmental conditions.


Assuntos
DNA Satélite , Tribolium , Animais , DNA Satélite/genética , Eucromatina , Regulação da Expressão Gênica , Heterocromatina , Humanos , Tribolium/genética
6.
Nat Cell Biol ; 23(8): 905-914, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34354237

RESUMO

Heterochromatin, typically marked by histone H3 trimethylation at lysine 9 (H3K9me3) or lysine 27 (H3K27me3), represses different protein-coding genes in different cells, as well as repetitive elements. The basis for locus specificity is unclear. Previously, we identified 172 proteins that are embedded in sonication-resistant heterochromatin (srHC) harbouring H3K9me3. Here, we investigate in humans how 97 of the H3K9me3-srHC proteins repress heterochromatic genes. We reveal four groups of srHC proteins that each repress many common genes and repeat elements. Two groups repress H3K9me3-embedded genes with different extents of flanking srHC, one group is specific for srHC genes with H3K9me3 and H3K27me3, and one group is specific for genes with srHC as the primary feature. We find that the enhancer of rudimentary homologue (ERH) is conserved from Schizosaccharomyces pombe in repressing meiotic genes and, in humans, now represses other lineage-specific genes and repeat elements. The study greatly expands our understanding of H3K9me3-based gene repression in vertebrates.


Assuntos
Proteínas Cromossômicas não Histona/fisiologia , Regulação da Expressão Gênica , Heterocromatina/fisiologia , Células Cultivadas , Sequência Conservada , Células Hep G2 , Histonas/metabolismo , Humanos
7.
Development ; 148(15)2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34345914

RESUMO

Sperm histones represent an essential part of the paternally transmitted epigenome, but uncertainty exists about the role of those remaining in non-coding and repetitive DNA. We therefore analyzed the genome-wide distribution of the heterochromatic marker H4K20me3 in human sperm and somatic (K562) cells. To specify the function of sperm histones, we compared all H4K20me3-containing and -free loci in the sperm genome. Sperm and somatic cells possessed a very similar H4K20me3 distribution: H4K20me3 peaks occurred mostly in distal intergenic regions and repetitive gene clusters (in particular genes encoding odorant-binding factors and zinc-finger antiviral proteins). In both cell types, H4K20me3 peaks were enriched in LINEs, ERVs, satellite DNA and low complexity repeats. In contrast, H4K20me3-free nucleosomes occurred more frequently in genic regions (in particular promoters, exons, 5'-UTR and 3'-UTR) and were enriched in genes encoding developmental factors (in particular transcription activators and repressors). H4K20me3-free nucleosomes were also detected in substantial quantities in distal intergenic regions and were enriched in SINEs. Thus, evidence suggests that paternally transmitted histones may have a dual purpose: maintenance and regulation of heterochromatin and guidance towards transcription of euchromatin.


Assuntos
Histonas/genética , Sequências Repetitivas de Ácido Nucleico/genética , Espermatozoides/fisiologia , Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Linhagem Celular Tumoral , DNA/genética , Éxons/genética , Genoma/genética , Heterocromatina/genética , Humanos , Células K562 , Masculino , Nucleossomos/genética , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/genética , Transcrição Genética/genética
8.
J Cell Sci ; 134(2)2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-34432033

RESUMO

Articular cartilage protects and lubricates joints for smooth motion and transmission of loads. Owing to its high water content, chondrocytes within the cartilage are exposed to high levels of hydrostatic pressure, which has been shown to promote chondrocyte identity through unknown mechanisms. Here, we investigate the effects of hydrostatic pressure on chondrocyte state and behavior, and discover that application of hydrostatic pressure promotes chondrocyte quiescence and prevents maturation towards the hypertrophic state. Mechanistically, hydrostatic pressure reduces the amount of trimethylated H3K9 (K3K9me3)-marked constitutive heterochromatin and concomitantly increases H3K27me3-marked facultative heterochromatin. Reduced levels of H3K9me3 attenuates expression of pre-hypertrophic genes, replication and transcription, thereby reducing replicative stress. Conversely, promoting replicative stress by inhibition of topoisomerase II decreases Sox9 expression, suggesting that it enhances chondrocyte maturation. Our results reveal how hydrostatic pressure triggers chromatin remodeling to impact cell fate and function. This article has an associated First Person interview with the first author of the paper.


Assuntos
Cartilagem Articular , Condrócitos , Diferenciação Celular , Heterocromatina , Humanos , Pressão Hidrostática
9.
Int J Mol Sci ; 22(14)2021 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-34299263

RESUMO

BACKGROUND: Charged-particle radiotherapy is an emerging treatment modality for radioresistant tumors. The enhanced effectiveness of high-energy particles (such as heavy ions) has been related to the spatial clustering of DNA lesions due to highly localized energy deposition. Here, DNA damage patterns induced by single and multiple carbon ions were analyzed in the nuclear chromatin environment by different high-resolution microscopy approaches. MATERIAL AND METHODS: Using the heavy-ion microbeam SNAKE, fibroblast monolayers were irradiated with defined numbers of carbon ions (1/10/100 ions per pulse, ipp) focused to micrometer-sized stripes or spots. Radiation-induced lesions were visualized as DNA damage foci (γH2AX, 53BP1) by conventional fluorescence and stimulated emission depletion (STED) microscopy. At micro- and nanoscale level, DNA double-strand breaks (DSBs) were visualized within their chromatin context by labeling the Ku heterodimer. Single and clustered pKu70-labeled DSBs were quantified in euchromatic and heterochromatic regions at 0.1 h, 5 h and 24 h post-IR by transmission electron microscopy (TEM). RESULTS: Increasing numbers of carbon ions per beam spot enhanced spatial clustering of DNA lesions and increased damage complexity with two or more DSBs in close proximity. This effect was detectable in euchromatin, but was much more pronounced in heterochromatin. Analyzing the dynamics of damage processing, our findings indicate that euchromatic DSBs were processed efficiently and repaired in a timely manner. In heterochromatin, by contrast, the number of clustered DSBs continuously increased further over the first hours following IR exposure, indicating the challenging task for the cell to process highly clustered DSBs appropriately. CONCLUSION: Increasing numbers of carbon ions applied to sub-nuclear chromatin regions enhanced the spatial clustering of DSBs and increased damage complexity, this being more pronounced in heterochromatic regions. Inefficient processing of clustered DSBs may explain the enhanced therapeutic efficacy of particle-based radiotherapy in cancer treatment.


Assuntos
Quebras de DNA de Cadeia Dupla/efeitos da radiação , DNA/efeitos da radiação , Radioterapia com Íons Pesados/efeitos adversos , Técnicas de Cultura de Células , Análise por Conglomerados , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , Eucromatina/genética , Eucromatina/efeitos da radiação , Fibroblastos , Radioterapia com Íons Pesados/métodos , Íons Pesados/efeitos adversos , Heterocromatina/genética , Heterocromatina/efeitos da radiação , Humanos , Autoantígeno Ku/genética , Autoantígeno Ku/efeitos da radiação , Transferência Linear de Energia/efeitos da radiação , Microscopia Eletrônica/métodos , Radiação Ionizante
10.
Nat Commun ; 12(1): 4359, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272378

RESUMO

Histone H3 lysine 9 (H3K9) methylation is a central epigenetic modification that defines heterochromatin from unicellular to multicellular organisms. In mammalian cells, H3K9 methylation can be catalyzed by at least six distinct SET domain enzymes: Suv39h1/Suv39h2, Eset1/Eset2 and G9a/Glp. We used mouse embryonic fibroblasts (MEFs) with a conditional mutation for Eset1 and introduced progressive deletions for the other SET domain genes by CRISPR/Cas9 technology. Compound mutant MEFs for all six SET domain lysine methyltransferase (KMT) genes lack all H3K9 methylation states, derepress nearly all families of repeat elements and display genomic instabilities. Strikingly, the 6KO H3K9 KMT MEF cells no longer maintain heterochromatin organization and have lost electron-dense heterochromatin. This is a compelling analysis of H3K9 methylation-deficient mammalian chromatin and reveals a definitive function for H3K9 methylation in protecting heterochromatin organization and genome integrity.


Assuntos
Fibroblastos/metabolismo , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Animais , Sistemas CRISPR-Cas , Sequenciamento de Cromatina por Imunoprecipitação , Cromatografia Líquida , Desmetilação , Epigênese Genética , Fibroblastos/enzimologia , Deleção de Genes , Heterocromatina/enzimologia , Heterocromatina/genética , Heterocromatina/ultraestrutura , Histona-Lisina N-Metiltransferase/genética , Hibridização in Situ Fluorescente , Espectrometria de Massas , Metilação , Camundongos , Microscopia Eletrônica de Transmissão , Mutação , Processamento de Proteína Pós-Traducional/genética , RNA-Seq , Sequências Repetitivas de Ácido Nucleico/genética , Retroelementos/genética , Transdução de Sinais/genética
11.
Int J Mol Sci ; 22(13)2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-34203193

RESUMO

Chromatin consists of a complex of DNA and histone proteins as its core components and plays an important role in both packaging DNA and regulating DNA metabolic pathways such as DNA replication, transcription, recombination, and chromosome segregation. Proper functioning of chromatin further involves a network of interactions among molecular complexes that modify chromatin structure and organization to affect the accessibility of DNA to transcription factors leading to the activation or repression of the transcription of target DNA loci. Based on its structure and compaction state, chromatin is categorized into euchromatin, heterochromatin, and centromeric chromatin. In this review, we discuss distinct chromatin factors and molecular complexes that constitute euchromatin-open chromatin structure associated with active transcription; heterochromatin-less accessible chromatin associated with silencing; centromeric chromatin-the site of spindle binding in chromosome segregation.


Assuntos
Centrômero/metabolismo , Eucromatina/metabolismo , Heterocromatina/metabolismo , Animais , Centrômero/genética , Cromatina/genética , Cromatina/metabolismo , Epigênese Genética/genética , Eucromatina/genética , Heterocromatina/genética , Humanos
12.
Nucleic Acids Res ; 49(13): 7406-7423, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34214177

RESUMO

Heterochromatin binding protein HP1ß plays an important role in chromatin organization and cell differentiation, however the underlying mechanisms remain unclear. Here, we generated HP1ß-/- embryonic stem cells and observed reduced heterochromatin clustering and impaired differentiation. We found that during stem cell differentiation, HP1ß is phosphorylated at serine 89 by CK2, which creates a binding site for the pluripotency regulator KAP1. This phosphorylation dependent sequestration of KAP1 in heterochromatin compartments causes a downregulation of pluripotency factors and triggers pluripotency exit. Accordingly, HP1ß-/- and phospho-mutant cells exhibited impaired differentiation, while ubiquitination-deficient KAP1-/- cells had the opposite phenotype with enhanced differentiation. These results suggest that KAP1 regulates pluripotency via its ubiquitination activity. We propose that the formation of subnuclear membraneless heterochromatin compartments may serve as a dynamic reservoir to trap or release cellular factors. The sequestration of essential regulators defines a novel and active role of heterochromatin in gene regulation and represents a dynamic mode of remote control to regulate cellular processes like cell fate decisions.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Células-Tronco Embrionárias/metabolismo , Heterocromatina/metabolismo , Proteína 28 com Motivo Tripartido/metabolismo , Animais , Caseína Quinase II/metabolismo , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/fisiologia , Cricetinae , Células-Tronco Embrionárias/citologia , Técnicas de Inativação de Genes , Humanos , Camundongos , Fosforilação , Serina/metabolismo , Proteína 28 com Motivo Tripartido/genética , Proteína 28 com Motivo Tripartido/fisiologia
13.
New Phytol ; 232(2): 835-852, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34289124

RESUMO

Despite a much higher proportion of intragenic heterochromatin-containing genes in crop genomes, the importance of intragenic heterochromatin in crop development remains unclear. Intragenic heterochromatin can be recognised by a protein complex, ASI1-AIPP1-EDM2 (AAE) complex, to regulate alternative polyadenylation. Here, we investigated the impact of rice ASI1 on global poly(A) site usage through poly(A) sequencing and ASI1-dependent regulation on rice development. We found that OsASI1 is essential for rice pollen development and flowering. OsASI1 dysfunction has an important impact on global poly(A) site usage, which is closely related to heterochromatin marks. Intriguingly, OsASI1 interacts with the intronic heterochromatin of OsXRNL, a nuclear XRN family exonuclease gene involved in the processing of an miRNA precursor, to promote the processing of full-length OsXRNL and regulate miRNA abundance. We found that OsASI1-mediated regulation of pollen development partially depends on OsXRNL. Finally, we characterised the rice AAE complex and its involvement in alternative polyadenylation and pollen development. Our findings help to elucidate an epigenetic mechanism governing miRNA abundance and rice development, and provide a valuable resource for studying the epigenetic mechanisms of many important processes in crops.


Assuntos
MicroRNAs , Oryza , Regulação da Expressão Gênica de Plantas , Heterocromatina/genética , MicroRNAs/genética , Oryza/genética , Pólen/genética , Poliadenilação
14.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi ; 33(3): 297-300, 2021 May 28.
Artigo em Chinês | MEDLINE | ID: mdl-34286533

RESUMO

OBJECTIVE: To investigate the karyotypes and C bands of Triatoma rubrofasciata in China, so as to understand its chromosome number, morphology and C-band staining of T. rubrofasciata. METHODS: The testis specimens were sampled from male T. rubrofasciata collected from Shunde City, Guangdong Province, prepared into slides of metaphase chromosomes and subjected to Giemsa staining and C-band staining. The morphology of metaphase chromosomes and the location of heterochromatin were observed using microscopy, and the long arm and short arm of each chromosome and total chromosome length were recorded to analyze the karyotypes and C bands of T. rubrofasciata. RESULTS: The male T. rubrofasciata presented a chromosome number of 2n = 25, including 22 autosomes and 3 sex chromosomes. The relative length of chromosomes ranged from 3.59% to 12.76%, the arm ratio was 1.06 to 1.24, and the centromere index was 44.76% to 48.47%. All chromosomes were metacentric chromosomes and the karyotype formula was 2n = 22 metacentric + X1X2Y, and the C bands varied on different chromosomes. No heterochromatin was found in the X chromosome, and the overall staining appeared pale, while heterochromatin was detected in all regions of the Y chromosome, and the overall staining appeared dark. In addition, heterochromatin was present in both ends of the autosome. CONCLUSIONS: The male T. rubrofasciata presents a chromosome number of 2n = 25 in China, and the karyotype formula is 2n = 22 metacentric + X1X2Y. C-banding shows dark staining of the Y chromosome, pale staining of the X chromosome, and dark staining of both ends of the autosome. Our data may provide insights into the investigation on the origin, evolution and gene mapping of T. rubrofasciata in China.


Assuntos
Triatoma , Animais , China , Heterocromatina , Cariótipo , Cariotipagem , Masculino , Triatoma/genética
15.
Biophys J ; 120(13): 2631-2643, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34087208

RESUMO

Within the nucleus of the eukaryotic cell, DNA is partitioned into domains of highly condensed, transcriptionally silent heterochromatin and less condensed, transcriptionally active euchromatin. Heterochromatin protein 1α (HP1α) is an architectural protein that establishes and maintains heterochromatin, ensuring genome fidelity and nuclear integrity. Although the mechanical effects of changes in the relative amount of euchromatin and heterochromatin brought about by inhibiting chromatin-modifying enzymes have been studied previously, here we measure how the material properties of the nuclei are modified after the knockdown of HP1α. These studies were inspired by the observation that poorly invasive MCF7 breast cancer cells become more invasive after knockdown of HP1α expression and that, indeed, in many solid tumors the loss of HP1α correlates with the onset of tumor cell invasion. Atomic force microscopy (AFM), optical tweezers (OT), and techniques based on micropipette aspiration (MA) were each used to characterize the mechanical properties of nuclei extracted from HP1α knockdown or matched control MCF7 cells. Using AFM or OT to locally indent nuclei, those extracted from MCF7 HP1α knockdown cells were found to have apparent Young's moduli that were significantly lower than nuclei from MCF7 control cells, consistent with previous studies that assert heterochromatin plays a major role in governing the mechanical response in such experiments. In contrast, results from pipette-based techniques in the spirit of MA, in which the whole nuclei were deformed and aspirated into a conical pipette, showed considerably less variation between HP1α knockdown and control, consistent with previous studies reporting that it is predominantly the lamins in the nuclear envelope that determine the mechanical response to large whole-cell deformations. The differences in chromatin organization observed by various microscopy techniques between the MCF7 control and HP1α knockdown nuclei correlate well with the results of our measured mechanical responses and our hypotheses regarding their origin.


Assuntos
Núcleo Celular , Proteínas Cromossômicas não Histona , Proteínas Cromossômicas não Histona/genética , Heterocromatina , Humanos , Células MCF-7 , Fatores de Transcrição
16.
Nat Commun ; 12(1): 3887, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162889

RESUMO

ATRX is a tumor suppressor that has been associated with protection from DNA replication stress, purportedly through resolution of difficult-to-replicate G-quadruplex (G4) DNA structures. While several studies demonstrate that loss of ATRX sensitizes cells to chemical stabilizers of G4 structures, the molecular function of ATRX at G4 regions during replication remains unknown. Here, we demonstrate that ATRX associates with a number of the MCM replication complex subunits and that loss of ATRX leads to G4 structure accumulation at newly synthesized DNA. We show that both the helicase domain of ATRX and its H3.3 chaperone function are required to protect cells from G4-induced replicative stress. Furthermore, these activities are upstream of heterochromatin formation mediated by the histone methyltransferase, ESET, which is the critical molecular event that protects cells from G4-mediated stress. In support, tumors carrying mutations in either ATRX or ESET show increased mutation burden at G4-enriched DNA sequences. Overall, our study provides new insights into mechanisms by which ATRX promotes genome stability with important implications for understanding impacts of its loss on human disease.


Assuntos
Replicação do DNA/genética , DNA/genética , Quadruplex G , Heterocromatina/genética , Proteína Nuclear Ligada ao X/genética , Células Cultivadas , Sequenciamento de Cromatina por Imunoprecipitação/métodos , DNA/química , DNA/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Instabilidade Genômica/genética , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Conformação de Ácido Nucleico , Proteína Nuclear Ligada ao X/metabolismo
17.
Nucleic Acids Res ; 49(13): 7389-7405, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34181735

RESUMO

A major stress response influenced by microRNAs (miRNAs) is senescence, a state of indefinite growth arrest triggered by sublethal cell damage. Here, through bioinformatic analysis and experimental validation, we identified miR-340-5p as a novel miRNA that foments cellular senescence. miR-340-5p was highly abundant in diverse senescence models, and miR-340-5p overexpression in proliferating cells rendered them senescent. Among the target mRNAs, miR-340-5p prominently reduced the levels of LBR mRNA, encoding lamin B receptor (LBR). Loss of LBR by ectopic overexpression of miR-340-5p derepressed heterochromatin in lamina-associated domains, promoting the expression of DNA repetitive elements characteristic of senescence. Importantly, overexpressing miR-340-5p enhanced cellular sensitivity to senolytic compounds, while antagonization of miR-340-5p reduced senescent cell markers and engendered resistance to senolytic-induced cell death. We propose that miR-340-5p can be exploited for removing senescent cells to restore tissue homeostasis and mitigate damage by senescent cells in pathologies of human aging.


Assuntos
Senescência Celular/genética , MicroRNAs/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Linhagem Celular , Proliferação de Células , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Regulação da Expressão Gênica , Heterocromatina , Humanos , Receptores Citoplasmáticos e Nucleares/metabolismo
18.
Biochim Biophys Acta Gene Regul Mech ; 1864(8): 194725, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34174495

RESUMO

The 3D spatial organization of the genome controls gene expression and cell functionality. Heterochromatin (HC), which is the densely compacted and largely silenced part of the chromatin, is the driver for the formation and maintenance of nuclear organization in the mammalian nucleus. It is functionally divided into highly compact constitutive heterochromatin (cHC) and transcriptionally poised facultative heterochromatin (fHC). Long regarded as a static structure, the highly dynamic nature of the heterochromatin is being slowly understood and studied. These changes in HC occur on various temporal scales during the cell cycle and differentiation processes. Most methods that capture information about the heterochromatin are static techniques that cannot provide a readout of how the HC organization evolves with time. The delineation of specific areas such as fHC are also rendered difficult due to its diffusive nature and lack of specific features. Another degree of complexity in characterizing changes in heterochromatin occurs due to the heterogeneity in the HC organization of individual cells, necessitating single cell studies. Overall, there is a need for live cell compatible tools that can stably track the heterochromatin as it undergoes re-organization. In this work, we present an approach to track cHC and fHC based on the epigenetic hallmarks associated with them. Unlike conventional immunostaining approaches, we use small recombinant protein probes that allow us to dynamically monitor the HC by binding to modifications specific to the cHC and fHC, such as H3K9me3, DNA methylation and H3K27me3. We demonstrate the use of the probes to follow the changes in HC induced by drug perturbations at the single cell level. We also use the probe sets combinatorically to simultaneously track chromatin regions enriched in two selected epigenetic modifications using a FRET based approach that enabled us tracking distinctive chromatin features in situ.


Assuntos
Epigênese Genética , Transferência Ressonante de Energia de Fluorescência , Heterocromatina/metabolismo , Metilação de DNA , Corantes Fluorescentes , Células HEK293 , Código das Histonas , Histonas/metabolismo , Humanos , Análise de Célula Única
19.
Microsc Microanal ; 27(4): 878-888, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34108070

RESUMO

A profound characteristic of field cancerization is alterations in chromatin packing. This study aimed to quantify these alterations using electron microscopy image analysis of buccal mucosa cells of laryngeal, esophageal, and lung cancer patients. Analysis was done on normal-appearing mucosa, believed to be within the cancerization field, and not tumor itself. Large-scale electron microscopy (nanotomy) images were acquired of cancer patients and controls. Within the nuclei, the chromatin packing of euchromatin and heterochromatin was characterized. Furthermore, the chromatin organization was quantified through chromatin packing density scaling. A significant difference was found between the cancer and control groups in the chromatin packing density scaling parameter for length scales below the optical diffraction limit (200 nm) in both the euchromatin (p = 0.002) and the heterochromatin (p = 0.006). The chromatin packing scaling analysis also indicated that the chromatin organization of cancer patients deviated significantly from the control group. They might allow for novel strategies for cancer risk stratification and diagnosis with high sensitivity. This could aid clinicians in personalizing screening strategies for high-risk patients and follow-up strategies for treated cancer patients.


Assuntos
Cromatina , Mucosa Bucal , Neoplasias Bucais , Eucromatina , Heterocromatina , Humanos , Microscopia Eletrônica , Mucosa Bucal/citologia , Neoplasias Bucais/diagnóstico
20.
PLoS Genet ; 17(6): e1009601, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34086674

RESUMO

Selection of C-terminal motifs participated in evolution of distinct histone H2A variants. Hybrid types of variants combining motifs from distinct H2A classes are extremely rare. This suggests that the proximity between the motif cases interferes with their function. We studied this question in flowering plants that evolved sporadically a hybrid H2A variant combining the SQ motif of H2A.X that participates in the DNA damage response with the KSPK motif of H2A.W that stabilizes heterochromatin. Our inventory of PTMs of H2A.W variants showed that in vivo the cell cycle-dependent kinase CDKA phosphorylates the KSPK motif of H2A.W but only in absence of an SQ motif. Phosphomimicry of KSPK prevented DNA damage response by the SQ motif of the hybrid H2A.W/X variant. In a synthetic yeast expressing the hybrid H2A.W/X variant, phosphorylation of KSPK prevented binding of the BRCT-domain protein Mdb1 to phosphorylated SQ and impaired response to DNA damage. Our findings illustrate that PTMs mediate interference between the function of H2A variant specific C-terminal motifs. Such interference could explain the mutual exclusion of motifs that led to evolution of H2A variants.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Histonas/genética , Processamento de Proteína Pós-Traducional , Schizosaccharomyces/genética , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Técnicas de Cultura de Células , Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Dano ao DNA , Evolução Molecular , Heterocromatina/química , Heterocromatina/metabolismo , Histonas/metabolismo , Fosforilação , Células Vegetais/metabolismo , Plantas Geneticamente Modificadas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transgenes
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